The specific enzymic cleavage of phosphoglyceroyl-ATP

Abstract

Rapid and extensive oscillations in rat heart adenine nucleotide content which could not be explained as metabolism of any known precursor, derivative or polymer caused us to propose the existence of some hitherto unrecognized derivative which exchanges rapidly with soluble nucleotides (for a review, see Lawson & Mowbray, 1986). A subsequent search after selectively labelling tissue purine nucleotides revealed a labile highly phosphorylated oligometric substance at specific activity equilibrium with ATP in both heart (Mowbray et a[., 1984) and kidney (Hutchinson et al., 1986b). Selective digestion allied to a variety of chromatographic techniques together with ” P nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry has suggested that it is a linear oligomer of ATP and 3-phosphoglycerate of the form ppp(5’A3’p3-glyceroyl1 -ppp), SfA3’p3-glycerate (Hutchinson et al., 19860). Work is currently in progress to confirm this structure by synthesizing the basic unit by a chemically unambiguous method. This report describes the recognition and partial purification from rat heart of an enzyme which degrades this oligomer in an AMP-dependent fashion. As with amino acid adenylates (Meister, 1963) and other mixed anhydrides of 5’-adenylic acid and a carboxylic acid (Jencks, 1963), phosphoglyceroyl-ATP (PG-ATP) is rapidly hydrolysed non-enzymically at neutral pH. However, we have found that PG-ATP is progressively stabilized in neutral solution as the concentration of Mn2+ or Mg2+ ions is increased to 2 mM; by 5 mwion, PG-ATP survives incubation at 37°C for 30 min without evidence of degradation (data not shown). Hence we routinely add 5-1Om~-MgC1, to all PG-ATP solutions. The [I4C]PG-ATP substrate is purified from perfused rat hearts as described by Hutchinson et al. (1986a). The assay used to survey cell fractions for their ability to degrade PG-ATP depends on the fact that the oligomer as purified is readily precipitated from 50% aqueous ethanol solution and can be trapped on a millipore (0.45pm) filter and assayed for radioactivity. The hydrolysed 8-[’4C]purine nucleotide remains in solution and passes through the filter. The standard conditions used in the assay are approx. 5 nmol of PG-ATP (350d.p.m.), 1 mM-AMP, 5 mM-MgCI,, 14 m~-2-mercaptoethanol, 50 mM-potassium phosphate, pH 7.0 in a total volume of 350~1 . After IOmin incubation at 37”C, 35Opl of ethanol is added and the mixture chilled at 20°C for 2 h before being filtered. The radioactivity on the filter and in the filtrate is assayed in 4ml of PCS scintillation mixture (Amersham International Ltd., Amersham, U.K.).